This invention relates to synthetic substrates and their uses in determining the presence of enzymes, and more particularly, to derivatives of 7-amino-4-trifluoromethyl-quinolone and process employing such derivatives to determine the presence of proteolytic enzymes.
The determination of specific enzymes and biological fluids including but not limited to blood, tissue homogenates, and protoplasm is very useful for the diagnosis of certain diseases.
Synthetic substrates have been developed and utilized for such determinations, resulting in clinical assay procedures having a high degree of specificity, reliability and sensitivity. Synthetic substrates have generally been amino acid derivatives of aromatic amines.
The number and arrangement of amino acids in the peptide moiety determines the enzyme specificity of a substrate. Any enzyme activity is measured by the amount of aromatic amine moiety liberated upon hydrolysis of a substrate. Generally, the process wherein an enzyme cleaves a substrate to liberate either a fluorophor or a chromophor is illustrated by the following: EQU substrate+enzyme.fwdarw.fluorophor+product
Exemplary synthetic substrates are disclosed in U.S. Pat. No. 3,862,011, dated Jan. 21, 1975, to Smith, and in U.S. Pat. No. 4,294,923, dated Oct. 13, 1981, to Smith et al.
Inherent in the reliability and sensitivity of clinical assay procedures employing synthetic substrates is the shift in the emission wavelengths between the synthetic substrate and the liberated fluorophor. Upon enzyme hydrolysis of the fluorogenic synthetic substrate, a shifting toward the red end of the emission wavelengths between the synthetic substrate and the liberated fluorophor occurs. The greater the shift, the greater the reliability of detecting the liberated fluorophor.
It would be an advancement in the art and highly desirable to develop a synthetic substrate which has a long wavelength shift, as well as possessing properties which would make it useful for the determination of specific enzymes and inhibitors in biological fluids.